Inorganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer

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Charlotte Lekieffre, Thierry Jauffrais, Emmanuelle Geslin, Bruno Jesus, Joan Bernhard, et al.. Inor- ganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer.

Scientific Reports, Nature Publishing Group, 2018, 8 (1), pp.10140. �10.1038/s41598-018-28455-1�.

�hal-02872797�

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Inorganic carbon and nitrogen assimilation in cellular

compartments of a benthic kleptoplastic foraminifer

Charlotte LeKieffre

, Thierry Jauffrais

, Emmanuelle Geslin

, Bruno Jesus

, Joan M. Bernhard

, Maria-Evangelia Giovani

& Anders Meibom

Haynesina germanica, an ubiquitous benthic foraminifer in intertidal mudflats, has the remarkable

ability to isolate, sequester, and use chloroplasts from microalgae. The photosynthetic functionality of these kleptoplasts has been demonstrated by measuring photosystem II quantum efficiency and

production rates, but the precise role of the kleptoplasts in foraminiferal metabolism is poorly understood. Thus, the mechanism and dynamics of C and N assimilation and translocation from the kleptoplasts to the foraminiferal host requires study. The objective of this study was to investigate, using correlated TEM and NanoSIMS imaging, the assimilation of inorganic C and N (here ammonium, NH

) in individuals of a kleptoplastic benthic foraminiferal species. H. germanica specimens were incubated for 20 h in artificial seawater enriched with H

NH

during a light/dark cycle. All specimens (n = 12) incorporated

C into their endoplasm stored primarily in the form of lipid droplets.

A control incubation in darkness resulted in no ¹³

C-uptake, strongly suggesting that photosynthesis is the process dominating inorganic C assimilation. Ammonium assimilation was observed both with and without light, with diffuse

N-enrichment throughout the cytoplasm and distinct

N-hotspots in fibrillar vesicles, electron-opaque bodies, tubulin paracrystals, bacterial associates, and, rarely and at moderate levels, in kleptoplasts. The latter observation might indicate that the kleptoplasts are involved in N assimilation. However, the higher N assimilation observed in the foraminiferal endoplasm incubated without light suggests that another cytoplasmic pathway is dominant, at least in darkness.

This study clearly shows the advantage provided by the kleptoplasts as an additional source of carbon and provides observations of ammonium uptake by the foraminiferal cell.

Kleptoplasty is defined as the process in which a cell sequesters algal chloroplasts while discarding or digesting other algal components

. This phenomenon is encountered in different organisms, such as sacoglossans (sea slugs)

, ciliates

, dinoflagellates

, and benthic foraminifera

.

Studies of benthic foraminiferal kleptoplasty have focused on shallow-water species inhabiting photic zones, especially Haynesina germanica and Elphidium spp. These studies have relied on ultrastructural observations and/or genetic analyses, which established the diatom origin of the kleptoplasts, or incubation/starvation exper- iments to define kleptoplast lifetimes and functionality once inside the foraminiferal cells

. Additionally

, showed that H. germanica and Elphidium williamsoni had a net uptake of inorganic carbon (H

CO

) in light, and experiments with oxygen microelectrodes demonstrated that maximal O

production by H. germanica depended on light intensity and light history

. A kleptoplastic strategy thus provides these organisms with both carbon

1Laboratory for Biological Geochemistry, School of Architecture, Civil and Environmental Engineering (ENAC), Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland. ²UMR CNRS 6112 LPG-BIAF, Université d’Angers, 2 Boulevard Lavoisier, 49045, Angers, CEDEX 1, France. ³EA2160, Laboratoire Mer Molécules Santé, Université de Nantes, Nantes, France. ⁴BioISI – Biosystems & Integrative Sciences Institute, Campo Grande University of Lisboa, Faculty of Sciences, Lisboa, Portugal. ⁵Woods Hole Oceanographic Institution, Department of Geology & Geophysics, Woods Hole, MA, USA. ⁶Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne, 1015, Lausanne, Switzerland. Correspondence and requests for materials should be addressed to C.L. (email: charlotte.lekieffre@epfl.ch)

Received: 18 April 2018 Accepted: 20 June 2018 Published: xx xx xxxx

OPEN

and a source of oxygen. Cesbron et al.

hypothesized that kleptoplasts might constitute an additional carbon source that may provide the kleptoplastic foraminifera a substantial competitive advantage, especially during periods of impoverished nutrients. However, the extent to which kleptoplasty contributes to the carbon within the foraminiferal cell storage via photosynthetic C assimilation has not been studied yet.

Foraminiferal kleptoplasts might also be involved in uptake of inorganic N. Indeed, diatoms, from which foraminifera sequester their kleptoplasts

, are able to assimilate ammonium through the chloroplast GS/

GOGAT (glutamate synthase and glutamine oxoglutarate aminotransferase) enzymatic pathway

. Kleptoplasty is also shown in deep-sea species

living in complete darkness and thus unable to perform photosynthe- sis

. Among these deep-sea species, Nonionella stella maintains kleptoplasts and associated functional enzy- matic machinery, including ribulose bis-phosphate carboxylase oxygenase (RuBisCO) and phosphoenol pyruvate carboxylase (PEP carboxylase), intact for months in the dark after sampling

. It was suggested that kleptoplasts in these deep-sea species are involved in assimilation of inorganic N

. Therefore, a similar role in shallow klepto- plastic foraminiferal species is possible and needs to be investigated.

To date, no studies have documented the timing and distribution of assimilation and translocation of C-photosynthates or N-compounds between kleptoplasts and the foraminiferal cell. To precisely trace the C and N assimilation within the different cell compartments in the kleptoplastic foraminiferal cell, the NanoSIMS (nanos- cale secondary ion mass spectrometry, a relatively recent ultra-high resolution isotopic imaging method

), was used in combination with transmission electron microscopy (TEM) and stable isotope labeling experiments

. This combined approach has already been successfully applied to study assimilation, storage, and transfer of C and N in several different marine organisms

, including foraminifera

. Using this integrative approach, the present study had three objectives: (1) investigate the role of kleptoplasts in C-fixation, (2) investigate the transfer and distribution of photosynthetically produced organic C within the host; and (3) investigate the potential role of kleptoplasts in foraminiferal N metabolism.

Results

H. germanica specimens were incubated for 20 h in artificial seawater enriched with 2 mM NaH

CO

and 10 μM

15

NH

Cl, following a light - dark cycle (Fig. 1, see details in Methods). Specimens were preserved for analysis at regular time intervals (i.e., after 4, 8, 12, and 20 hours).

TEM observations of foraminiferal cytoplasm. The cytoplasm of all specimens had well-preserved ultrastructure (Fig. 2A), as well as intact mitochondria with visible double-membranes and cristae (Fig. 2B).

Numerous small lipid droplets (diameter of ca. 500 nm), recognized by their waxy appearance, were observed in the cytoplasm (Fig. 2C), along with some larger lipid droplets ranging from 1 to 3 µm in diameter. Numerous small oval fibrillar vesicles (ca. 500 nm in length), with the fibrils arranged in parallel, and spherical to oval-shaped electron-opaque bodies (200–500 nm) were observed in the cytoplasm (Fig. 2D,E), along with occasional tubulin paracrystals identifiable due to the regular pattern of their ultrastructure revealed by high-magnification TEM imaging (Fig. 2F). In all specimens, we observed many small structures (2 to 3 µm in length) variable in shape but mainly ovoid (Fig. 2A) with the presence of numerous vacuoles within their matrix (Fig. 2G,H). Henceforth, we refer to these as “multi-vacuolar structures”.

In all observed specimens, TEM images of the endoplasm revealed well-preserved kleptoplasts with visible pyrenoids and thylakoids (Figs 2A, 3, 4). These kleptoplasts ranged in size from 2 to 10 µm in diameter. Generally, their outlines were circular to oval. They were distributed in the endoplasm with no clear pattern and often sur- rounded by an electron-lucent space between the kleptoplast membranes and the endoplasm. Some of the small lipid droplets were observed adjacent to the kleptoplast periphery. In some cases, lipid droplets were even closely associated with kleptoplast membranes (Fig. 3B).

Uptake of H

CO

within foraminiferal cells. In Experiment 1, starting at t

8 h,

C-enrichments

were detected in all specimens. The signal was concentrated in fibrillar vesicles, electron opaque bodies, and

lipids (Figs 4, 5A,B,C). In contrast, only one specimen from the first time point (i.e. at 4 h) exhibited

C-enriched

structures, concentrated in fibrillar vesicles and electron opaque bodies (Fig. 4). Although some lipid drop-

lets were present, they were only slightly enriched at 4 h (Figs 4 and 6). All specimens collected between 8

Figure 1. Schematic of Experiments 1 and 2, exposing H. germanica to different light conditions. Three

specimens were sampled at each indicated time point. See text for details.

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and 20 h of incubation exhibited

C-enrichments in the endoplasm. The

C-enrichment (expressed in

C) of electron-opaque bodies significantly increased during the light phase from ca. 40‰ at 4 h to 180‰ at 8 h (p

0.05) and remained stable through the dark phase of the experiment, i.e. between 8 and 20 h of incuba- tion (Fig. 6B). The

C-enrichments of the fibrillar vesicles and the lipid droplets were relatively stable during the incubation at ca. 200 to 300‰ and 60 to 150‰, respectively (Fig. 6C,D). The cytoplasm itself was slightly more enriched after 8 h of incubation than after 4 h, with averages of ca. 100‰ and 40‰, respectively (Fig. 6A).

However, the cytoplasmic enrichment did not change statistically between 8 h and 20 h (p

0.05; Fig. 6A). No

13

C-enrichments were found in foraminifera incubated with H

CO

in darkness (Experiment 2; Figs 6 and 7).

Uptake of

NH

in the foraminiferal cell. All specimens of Experiment 1 exhibited detectable

15

N-enrichments. In the cytoplasm of H. germanica,

N-enrichments significantly increased between 4 and 8 h Figure 2. TEM micrographs of the cytoplasm and organelles of Haynesina germanica. (A) Aspect of the cytoplasm in a chamber of the penultimate whorl. (B) Intact mitochondria with well-defined cristae and intact double-membranes. (C) Small lipid droplets. (D) Fibrillar vesicles. (E) Electron-opaque bodies, (F) Tubulin paracrystals; Inset: higher magnification revealing regular pattern of the paracrystal ultrastructural organization. (G,H) multi-vacuolar structures. Arrowheads: multi-vacuolar structures; c: chloroplast; eo:

electron-opaque bodies, fv: fibrillar vesicles, li: lipid droplets; m: mitochondria, tp: tubulin paracrystals, v-

vacuole. Scale bars: A: 2 µm; B, inset F: 200 nm; C–H: 500 nm.

(during the light phase), from ca. 250 to 550‰ (p < 0.05), and stabilized between 8 and 20 h (p > 0.05), i.e., during the dark phase (Fig. 6A). Similar to the observed

C-enrichments, the

N-signal was concentrated in electron-opaque bodies and fibrillar vesicles (Figs 4, 5A,B and 6). The

N-enrichment of electron-opaque bod- ies was relatively stable from 4 to 12 h (between 1800–2900‰) and then decreased to less than 1000‰ at 20 h (p < 0.05 between 4 and 20 h; Fig. 6B). At the same time, the

N-enrichment of the fibrillar vesicles increased throughout the incubation passing from ca. 425‰ at 4 h to 1320‰ after 20 h (p < 0.05). Some of these organelles were occasionally enriched in

N but not in

C. The tubulin paracrystals and the multi-vacuolar structures were also strongly enriched in

N after 8 h (Fig. 5D,E). Kleptoplasts rarely exhibited

N-enrichments, and if such enrichments were observed, they were always moderate to low (Fig. 5F).

In Experiment 2, after 8 h in darkness, the foraminifera had incorporated a much higher concentra- tion of

NH

(Fig. 6) compared with Experiment 1 at any given time (p < 0.05); the cytoplasmic average

15

N-enrichment reached a value of ca. 1100‰ after 8 h of incubation in darkness. The fibrillar vesicles were also significantly more

N-enriched during the second experiment reaching values of ca. 2400‰ (vs. maximum of ca. 1300‰ during Experiment 1). In contrast, the electron-opaque bodies exhibited

N-enrichment compara- ble to those recorded during the first experiment, i.e. around ca. 2600‰. As in Experiment 1, the

N isotopic signal was observed most concentrated in electron-opaque bodies, fibrillar vesicles, tubulin paracrystals, in the multi-vacuolar structures, as well as in a few kleptoplasts (Figs 6 and 7).

Discussion

All specimens exhibited mitochondria with intact cristae and double-membranes indicating that they were alive at the time of fixation

. The kleptoplasts observed in our study correspond to the morphological description of

for H. germanica collected from the Bourgneuf Bay (as in this study) and from the Wadden Sea (Mokbaai, NL). Specimens were well preserved with undamaged thylakoids and pyrenoids. The electron-lucent space that sometimes surrounded the kleptoplasts was also previously described by

and ascribed to a possible fixation artefact. Indeed, TEM observations of the same species fixed using high pressure freezing and freeze substitution Figure 3. TEM micrographs of one chloroplast in Haynesina germanica cytoplasm. (A) Intact pyrenoid and thylakoids. (B) Higher magnification image showing two small lipid droplets in contact with the chloroplast membranes. The chloroplast membranes adjacent to the lipid vesicle are disrupted. li: lipid droplets, py:

pyrenoid, th: thylakoid. Scale bars: A: 2 µm; B: 500 nm.

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instead of “classic” chemical fixation revealed kleptoplasts without electron-lucent space, with their membranes directly in contact with the surrounding foraminiferal cytoplasm

.

The paired TEM-NanoSIMS observations allowed the visualization of inorganic C uptake (H

CO

) within foraminiferal cells incubated under a light/dark cycle (Figs 4 and 6). The absence of

C assimilation in contin- uous darkness (Experiment 2, Figs 6 and 7) and the observed production of O

under light, as observed for H.

germanica in other studies

, strongly suggests that H. germanica kleptoplasts have a functional Calvin-Benson

cycle, resulting in the production and transfer of

C-photosynthates to the H. germanica cell. Foraminifera can

acquire C by different trophic mechanisms

, but they are not known to actively uptake inorganic C in the absence

of either bacterial or algal symbionts or in the presence of kleptoplasts. We found no indications of the pres-

ence of prokaryotic symbiotic photosynthetic organisms and, therefore, suggest that the observed incorpora-

tion of

C-bicarbonate is the result of photosynthesis occurring in the kleptoplasts. However, the absence of

Figure 4. Time-evolution of

C and

N uptake and localization within the cytoplasm of H. germanica during

Experiment 1 (light/dark incubation with H

CO

and

NH

). Left column: TEM micrographs. Middle and

right columns: corresponding NanoSIMS

C and

N images, respectively, expressed in ‰. Arrows: fibrillar

vesicles; arrowheads: electron opaque bodies; circles and li: lipid droplets, white triangles: multi-vacuolar

structures; c: chloroplast; tp: tubulin paracrystals. Scale bars: 2 µm.

13

C-enrichment inside the kleptoplasts (Figs 4 and 5) was unexpected. This absence of

C-enrichment can be attributed to the fact that the

C-photosynthates are quickly transported away from the kleptoplasts and thus the

C-enrichment stay below the detection limit of the NanoSIMS. This hypothesis is supported by previous NanoSIMS studies of autotrophic

C-exchanges in the symbiotic association between dinoflagellates and corals, where

C-enrichments in dinoflagellate chloroplasts were systematically much lower than in other sub-cellular organelles

. Additionally, studies on H. germanica have shown that the cellular machinery necessary for chloro- plast maintenance is unlikely to be functional

, which could explain why, in our Experiment 1, the kleptoplasts did not accumulate

C within their structures. In summary, our observations show that kleptoplasts in H. ger- manica are able to assimilate inorganic C and form

C-photosynthates that are transferred to the host cell, but the kleptoplasts do not themselves become enriched in

C (above the detection limit of the NanoSIMS).

The numerous multi-vacuolar structures observed in H. germanica (Fig. 2A,G,H) are somewhat similar to the bacteria observed in another benthic species, Globocassidulina cf. G. biora

. The presence of numerous such Figure 5. Foraminiferal organelles enriched in

C and/or

N in Experiment 1 at different time points. Left column: TEM micrographs. Middle and right columns: corresponding NanoSIMS δ

C and δ

N images, respectively, expressed in ‰. (A) electron-opaque bodies (after 8 h of incubation), (B) fibrillar vesicles (after 12 h of incubation), (C) lipid droplets (after 8 h of incubation), (D) tubulin paracrystals (after 20 h of incubation), (E) multi-vacuolar structures (after 12 h of incubation), (F) chloroplasts (after 8 h of incubation).

Arrowheads: electron-opaque bodies; arrows: fibrillar vesicles; circles and li: lipid droplets; c: chloroplasts; white

triangles: multi-vacuolar structures; c: chloroplast; tp: tubulin paracrystals. Scale bars: 2

m.

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vacuoles within prokaryotic cells has been described and linked to different biological functions, such as buoy- ancy (gas vacuoles) in planktonic bacteria or nitrate vacuoles in filamentous sulfur bacteria

. Thus these struc- tures could potentially be interpreted as endobionts. Is it noteworthy that they were not labeled in

C (Fig. 5E).

Other NanoSIMS studies looking at

C-bicarbonate assimilation in cyanobacteria, anaerobic photosynthetic bacteria or chemotrophic bacteria have shown strong bacterial

C-enrichments

. Therefore, even if the multi-vacuolar structures observed in our study were bacteria, the absence of

C incorporation into their struc- ture suggests that they are not photosynthesizing or assimilating inorganic carbon and that thus they do not play any role in the inorganic

C assimilation in Haynesina germanica.

Carbon was assimilated during the light phase, transferred to the foraminiferal cell, and accumulated in spe- cific organelles: electron-opaque bodies, fibrillar vesicles, and lipid droplets. The

C-assimilation dynamics in Figure 6. Average

C (black bars) and

N (white bars) enrichment of (A) the cytoplasm, (B) electron-opaque bodies, (C) fibrillar vesicles and (D) lipid droplets of H. germanica (n

3) in Experiments 1 and 2. Error bars represent one standard deviation. Red lines indicate natural variations in

C (solid lines) and

N (dotted lines) enrichments as measured by NanoSIMS in similar areas of unlabelled control H. germanica specimens (n

3;

δ¹³

C

0

40‰, and

N

0

60‰, 3σ).

electron-opaque bodies and fibrillar vesicles should be interpreted with caution, as these organelles are poorly understood

. Indeed, we do not know their function(s) in the cell, how fast they are produced, their turn over and what triggers their production. In addition, we do not know if the

C-enriched hotspots observed within the foraminiferal endoplasm correspond only to newly formed electron-opaque bodies and fibrillar vesicles, or if

C-enriched material was added to pre-existing organelles; which is likely considering the high variability observed for enrichment values (Fig. 6). However, we note that none of these organelles exhibited an increase in their

C-enrichment during the dark phase of Experiment 1, and none of the analyzed organelles showed

13

C-enrichment during Experiment 2. This strongly indicates that there are no cytoplasmic foraminiferal path- ways for inorganic carbon assimilation; i.e. carbon is assimilated only via the photosynthetic kleptoplasts.

Lipid droplets are considered to be the main C storage form in foraminifera

. A similar accumulation process/

sequence has been observed in the symbiotic planktonic foraminifer Orbulina universa, where photosynthesis led to an assimilation of inorganic C (H

CO

) stored in the form of lipid droplets

. In kleptoplastic sea slugs (e.g., Elysia chlorotica), lipid droplets observed in the animal tissue where argued to result from the exudation of lipids from the plastids because their fatty acids had a large proportion of algal-derived eicosapentaenoic acid (20:5)

. However, these authors could not determine whether the plastids transferred fatty acids directly via triacylglycerols (TAGs), or as free fatty acids that may be further transformed by the host into lipid droplets. The de novo produc- tion of triacylglycerol by chloroplasts in marine algae has been demonstrated

. Furthermore, de novo fatty acid synthesis is known to occur in plant cell chloroplasts

, followed by a transfer in the form of free fatty acids to the cytosol

. Additional transfer of soluble molecules such as maltose or glucose across the chloroplast membranes through transporters also occurs in plant cells

. The close spatial association between kleptoplast membranes and small lipid droplets observed here (Fig. 3) may indicate a potential transfer of C via exudation of small lipid droplets from kleptoplast to the H. germanica cell, although the detailed mechanisms by which the fatty acids cross kleptoplast membranes remain unknown. Unfortunately, the distribution of soluble molecules cannot be investi- gated with NanoSIMS because the sample preparation protocol causes near complete loss of such components

.

Teugels et al.

reported that ammonium assimilation by the kleptoplastic sacoglossan Elysia viridis was significantly higher during light exposure than in darkness. This is consistent with the glutamine oxoglutarate aminotransferase (GOGAT) enzyme pathway that requires electron donors (e.g., reduced ferredoxin) formed during photosynthetic electron transport

. Furthermore, the glutamate synthase (GS) metabolic reaction is ATP-dependent, and gene expression of some key enzymes (GS and GOGAT) is light regulated

. In corals, sym- biotic dinoflagellate GS/GOGAT enzymes are thought to be the main ammonium assimilation pathway

. Furthermore, cnidarian cells are also known to produce cytosol glutamate dehydrogenase (GDH)

, which has a dual function: 1) it converts glutamate to

-ketoglutarate and ammonium that is subsequently assimi- lated into chloroplasts via the GOGAT pathway

; 2) it catalyzes the opposite reaction, i.e. the amination of the

-ketoglutarate to produce the amino acid glutamate

.

In our study, the observation of

N-labeled kleptoplasts in H. germanica incubated both in light and in darkness seems consistent with a GS/GOGAT kleptoplastic pathway for ammonium assimilation (Fig. 5H). However, the uptake of

N-ammonium was higher after 8 h of incubation in total darkness (Experiment 2) than in Experiment 1 (light-dark cycle) (Fig. 6). This higher uptake in darkness compared to light is inconsistent with the light regu- lation of the GS/GOGAT enzymatic machinery

. Ammonium incorporation might, thus, also occur by another N-assimilation pathway in foraminifera, for example, through the glutamine dehydrogenase (GDH) pathway.

Figure 7.

C and

N uptake and localization within the cytoplasm of H. germanica during Experiment 2

(continuous dark incubation with H

CO

and

NH

). Left column: TEM micrographs. Middle and right

columns: corresponding NanoSIMS

C and

N images, respectively, expressed in ‰. Arrows: fibrillar

vesicles; arrowheads: electron opaque dense bodies; circles: lipid droplets, white triangles: multi-vacuolar

structures; c: chloroplast, tp: tubulin paracrystals. Scale bars: 2 µm.

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The same organelles (fibrillar vesicles, electron-opaque bodies and multi-vacuolar structures) were found to be

N-enriched in both Experiments 1 and 2. As previously noted for

C-assimilation, the

N-assimilation dynamics in electron-opaque bodies and fibrillar vesicles is difficult to interpret, due to the lack of knowledge about the function of these organelles

. However, the electron-opaque bodies and the fibrillar vesicles seem to have different patterns for

N assimilation. While the electron-opaque bodies incorporated large amounts of

N even after only 4 hours and then gradually lost this

N-enrichment over time, the fibrillar vesicles assimilated

N throughout the 20 h incubation, independent of light condition. It is noteworthy that the

N-assimilation pattern clearly differs from the

C-assimilation dynamics in both organelle types.

Finally, ammonium is known to be a suitable N source for many marine prokaryotes

. Thus, if the multi-vacuolar structures, abundant in all H. germanica specimens (Fig. 2A,G,H) are endosymbionts (see above) they would be expected to incorporate

NH

, as is indeed observed (Fig. 5G). They could, thus, constitute another putative nitrogen assimilation pathway for the benthic foraminifer H. germanica. However, we cannot conclude further in this study about the symbiotic nature of these putative prokaryotes in H. germanica, nor about their role in foraminiferal N metabolism.

A comparative study of organic C (algae) uptake through feeding between the two dominant foraminiferal species inhabiting mudflats, the akleptoplastic Ammonia sp. and kleptoplastic H. germanica, showed a higher uptake rate by the former

. Our results highlight that H. germanica can fix inorganic carbon. Therefore, unlike Ammonia sp., H. germanica does not rely solely on heterotrophy to meet its C requirements. The mixotrophic feeding strategy of H. germanica might give a competitive advantage and allow it to become the dominant foraminifera in mudflat environments

. In addition, whether H. germanica assimilates nitrogen through the kleptoplasts, potential endosymbionts, and/or by another pathway specific to foraminifera, our observations demonstrate that it is also capable of using inorganic N as a nutrient source. Further investigation is required to quantify this uptake and elucidate the role of this benthic foraminifera in the N cycle, especially since H. german- ica thrives in coastal ecosystems that are subject to increasing eutrophication

.

Conclusion

Our study demonstrates inorganic C assimilation in H. germanica, most likely via the kleptoplasts. The absence of

C assimilation in darkness combined with previous studies documenting O

production in light strongly suggest that photosynthesis is the process dominating inorganic C-assimilation in this species. Subsequently, photosynthates are transferred to the foraminiferal cell and utilized for its metabolism. Thus, these observations clearly show the role played by the kleptoplasts in H. germanica carbon metabolism, providing the foraminiferal cell with an additional autotrophic source of C. The observation of small lipid droplets attached to the klepto- plast membranes may suggest a transfer of C from the kleptoplasts to the foraminiferal cell in the form of lipids.

However, the detailed mechanism(s) involved in this C transfer remains unknown. The kleptoplasts may also pro- vide additional N sources to foraminiferal metabolic pathways via GS-GOGAT enzymes. However, ammonium assimilation was more efficient in darkness, requiring the existence of other N-assimilation pathways.

Material and Methods

Experiment 1: light/dark cycle incubation with H

CO

and

NH

. Living foraminifera were collected on April 9, 2015, at low tide on the intertidal mudflat of the Bourgneuf Bay (France, 47°00

59.4

N 2°01

29.8

W). The top centimeter of the sediment was sampled, sieved over a mesh of 150

m with in situ seawa- ter and the

150

m fraction was immediately transported in the dark over

3 hours to the laboratory in Nantes.

In the laboratory, healthy living individuals of H. germanica were selected under a binocular microscope based on their cytoplasm color (i.e. yellow-brownish material spread through all the chambers of the specimen, except the youngest chamber). The selected specimens were placed into 5 Petri dishes (5 specimens per Petri dish) filled with artificial seawater (ASW, Red Sea Salt, salinity

35, pH

8.0). Four of the Petri dishes contained ASW enriched with 2 mM NaH

CO

and 10

M

NH

Cl. The fifth Petri dish contained isotopically normal artificial seawater of the same chemical composition: specimens from this dish were fixed at T0 (beginning of the experiment) and served as controls for NanoSIMS analysis (see below). All other Petri dishes were placed in an incubator (Fytoscope FS130, temperature: 18 °C, light intensity: 90

mol m

s

). After 8 h of light exposure they were transferred to dark conditions for another 12 h. Except for the control specimens, the foraminifera remained in the spiked ASW throughout the experiment. At each time point, i.e., after 4, 8, 12, and 20 hours, one Petri dish was removed from the incubator (Fig. 1) and the 5 specimens contained in this Petri dish were immediately chemically fixed.

Experiment 2: Incubation in continuous darkness with H

CO

and

NH

. H. germanica speci- mens were collected on May 16, 2015, at low tide on the intertidal mudflat of the Bourgneuf Bay (France) follow- ing the same procedure as described above. Five living specimens were selected and placed in a Petri dish with artificial seawater (Red Sea Salt, salinity

35; pH

8.0) enriched with 2 mM NaH

CO

and 10

M of

NH

Cl.

They were incubated in continuous darkness for 8 h (Fig. 1) and immediately chemically fixed at the end of this incubation. Control samples, which were incubated in normal seawater, where fixed at the beginning of the experiment (T0; Fig. 1).

Preparation for TEM-NanoSIMS analysis. The specimens were chemically fixed following the pro-

tocol described

). Briefly, foraminifera were fixed immediately after removal from the incubator, with a mix

of 4% glutaraldehyde and 2% paraformaldehyde diluted in 0.1 M cacodylate buffer, 0.4 M sucrose, and 0.1 M

NaCl (pH

7.4) at room temperature for 24 h. They were then stored at 4 °C until further processing. Further

chemical processing and transmission electron microscope (TEM) imaging of the foraminifera were performed

at the Electron Microscopy Facility of the University of Lausanne (Switzerland). After rinsing, specimens were

Foraminiferal sections were imaged with the NanoSIMS ion microprobe with a 16 keV primary ion beam of Cs

focused to a beam spot of ca. 100–150 nm. The secondary molecular ions

C

,

C

C

,

C

N

and

12

C

N

were collected simultaneously in electron multiplier detectors at a mass-resolution of ca. 10000, enough to resolve potential interferences in the mass spectrum

. Isotopic image dimensions ranged from 15

15 µm to 30

30 µm with 256

256 pixel resolution. For each image, 6 layers were acquired, drift corrected and super- imposed using the software L’IMAGE (developed by Dr. Larry Nittler, Carnegie Institution of Washington DC, USA). Quantified

C/

C and

N/

N ratios were obtained as follows:

C (‰) (( C

/ C

) 1) 10

13 3

δ = − ×

δ¹⁵

N (‰)

(( N

/ N

)

1)

10

where C

is the measured

C

C

/

C

ratio of the sample and C

is the average

C

C

/

C

ratio measured in unlabeled samples (control). Similarly, N

is the measured

C

N

/

C

N

ratio of the sample and N

is the average

C

N

/

C

N

ratio measured in unlabeled samples. The software Look@NanoSIMS

was used to determine the isotopic enrichment of specific organelles that were identified morphologically from TEM images.

Regions of interest (ROIs) to quantify the average isotopic enrichment of the organelles were defined from the TEM images previously aligned with the NanoSIMS images (based on the

C

N image). For the average iso- topic enrichment of the cytoplasm, three circles of ca. 2 μm in diameter were drawn per image, avoiding highly

15

N-enriched organelles. For each specimen, between one and three NanoSIMS images were analyzed.

Statistical analysis. For each time point, three specimens were analyzed.

C and

N values for main orga- nelles and cytoplasm were obtained by calculating the average of ROIs within each specimen, and then calculating the average of the three specimens for each time point. The error bars provided are thus standard deviations rep- resenting inter-specimen variability. However, for the statistical analysis, a linear mixed-effects (LME) model was constructed using all the ROIs of the three specimens for each time point (taking into account pseudo-replication effects, i.e. regrouping ROIs from three different specimens into one category), followed by a Tukey multiple comparison test. The results of the Tukey multiple comparisons tests are given in the Supplementary Data S1. The statistical analyses were performed with Rstudio software using a significance level set at α

0.05.

Data Availability. The datasets generated and/or analyzed during the current study are available in the PANGAEA Repository (https://doi.org/10.1594/PANGAEA.891407 data will be published upon acceptance of the manuscript).

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Acknowledgements

The electron microscopy platform at the University of Lausanne (Switzerland) is thanked for technical assistance.

This work was supported by the Swiss National Science Foundation (grant no. 200021_149333) and was part of the CNRS EC2CO-Lefe project ForChlo. It was also supported by the Region Pays de la Loire (Post-doc position of TJ, on FRESCO project) as well as the WHOI Robert W. Morse Chair for Excellence in Oceanography and The Investment in Science Fund at WHOI.

Author Contributions

C.L., M.E.G. and E.G. collected the samples from the field. C.L., E.G. and A.M. designed the experiment. C.L. and M.E.G. analysed the samples. C.L., T.J., E.G., B.J., J.M.B. and A.M. interpreted the data and discuss the results.

C.L. performed the statistical analysis, wrote the manuscript and prepared the figures. All authors reviewed the manuscript.

Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-28455-1.

Competing Interests: The authors declare no competing interests.

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